The phenomenon of the symbiotic relationship between legumes and the Rhizobium bacteria which nodulate their roots is widely known. The bacteria, living in the nodules in the legume roots, fix nitrogen directly from the atmospheric nitrogen thereby converting nitrogen to biologically useful nitrogenous compounds. This process not only provides nitrogen to the plant for protein synthesis, but also enriches the soil in which the legume is grown by leaving nitrogenous nutrients in the soil for later crops. Examples of legumes which are capable of symbiotic relationship with Rhizobium bacteria are peas, beans, alfalfa, red clover, white clover, vetch, and lupines.
Since other agriculturally important plants are unable, either directly or indirectly, to fix nitrogen directly from the air, and thus generally are dependent upon nitrogenous fertilizers to introduce sufficient nitrogen content in the soil for good yield, it is quite common for legumes to be alternated each growing season with non-legume crops in important agricultural areas to facilitate economical enhancement of nitrogen content in the soil. This practice, known as crop rotation, is wide spread Furthermore, some legumes, and in particular soybeans, peas, beans, and alfalfa, are important commercial crops in their own right and growth of these crop plants is greatly facilitated by an ample supply of combined or fixed nitrogen.
There are a number of species of Rhizobium bacteria and different Rhizobium species are adapted to form symbiotic relationships, and nodules, only in the roots of specific legumes. For example, R. japonicum nodulates the roots of soybeans, R. trifolii nodulates the roots of clovers, R. meliloti grows symbiotically with alfalfa and sweet clovers, R. leguminosarum is used with peas and vetches, and R. phaseoli nodulates the roots of common garden beans. Therefore, in commercial agricultural practices, it is quite a common practice to inoculate the soil or the seeds of the legumes with a culture of the appropriate Rhizobium species. This inoculant is commonly done by coating the seeds before planting, dusting planted seeds, or by spreading the inoculant in the furrows of the planted legume seeds.
In creating and formulating a Rhizobium inoculant, it is desirable that the Rhizobium strain contained in the inoculant have an enhanced ability to fix nitrogen for the benefit of the plant and for soil conditioning. Therefore, much research has logically been conducted on methods for improving the nitrogen fixation of Rhizobium species. Most of the currently commercially available Rhizobium inoculants include strains having high nitrogen fixing ability selected from natural populations.
However, the ability to fix nitrogen does not ensure the survivability of the bacteria in field conditions. Because legumes have been cultivated for many generations in most of the important agricultural areas of the world, many strains of Rhizobium bacteria now freely live indigenously in important agricultural areas. The bacteria may not have been naturally indigenous to all these areas, but introduced strains have populated many of these areas and since they survive freely they may be considered effectively indigenous.
All free-living bacteria, in the soil or elsewhere, are continually subject to environmental pressures, and naturally tend to mutate and be selected by the pressures of the ecological niche which they occupy to be further adapted and competitive for that niche. The indigenous Rhizobium strains are, of course, subject to these pressures. Therefore many of the soils in agricultural areas now contain indigenous Rhizobium strains which have evolved to be adapted for survival in competitive existence in that particular soil environment. Accordingly, even if a farmer introduces into a field a Rhizobium inoculant strain having high nitrogen fixing capability, there is no guarantee that the introduced Rhizobium strain will predominate the roots of the legume plants. The introduced inoculant strain promptly enters into ecological competition with the indigenous strains already present in the soil to nodulate the plants, and, many times, the indigenous populations are an advantage because they are previously selected by ecological pressures for competition in precisely the environment of that field or climatic area. Accordingly, even a high nitrogen fixing Rhizobium strain which can effectively nodulate the legume and fix nitrogen effectively without competition may be an ineffective inoculant in the field since the introduced strain may not actually populate the roots of the legumes, but may be overwhelmed by a locally indigenous strain, which may not have optimal nitrogen fixation abilities. No prior teachings are known on either the desirability of or the process for creating novel Rhizobium strains which are both competitive in field conditions and have high nitrogen fixation abilities.
It has been known previously that legume root exudate can be used to cause Rhizobium cells to develop more lectin-binding sites. It is also known that such exudates can be used as a pretreatment to foster and to enhance the ability of Rhizobium to nodulate when inoculated onto a legume plant.